We encountered a problem for testing protein protein interaction by Y2H assay. The AH109 transformants carrying empty pGBK and pGAD vectors can still survive on triple SD medium (-Leu-Trp-His) without 3AT supplement. X-beta-gal and ONPG assay indicated that the negative control also had lacZ activity. I was wondering if anyone has ever met the same situation or can give suggestions. Thanks.
Hi there,
AH109 strain should not grow without histidine unless there is a leaky expression of HIS3 gene which is under GAL4 responsive elements (in presence of 3AT the strain should be his-). As LacZ is also under GAL4 responsive elements in AH109, it explains why you get leaky lacZ activity (just as for HIS3 in absence of 3AT). The main issue to use this strain for X-gal assay is to check that in presence of interacting partners the lacZ activity is significantly increased.
FYI: we tested several independent AH109 strains from different labs and found all the strains (kinda 'wild-type', eg. not carry pGBK-T7 and pGAD-T7) can be stained by x-gal, though the LacZ activity was variate. Therefore, I strongly suggest to avoid AH109 if you want to do X-gal assay. We also confirmed Y187 is okay for X-gay assay.
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